Abstract Pulmonary arterial hypertension (PAH) is a disease of pulmonary vasculature with a high mortality rate of up to 45% three years after diagnosis. PAH is often associated with the loss of endothelium-dependent vasodilation, which has long been thought to be a major contributor to development of PAH. However, the underlying mechanisms for the loss of endothelium-dependent vasodilation in PAH remain unclear, particularly in the native endothelium from small pulmonary arteries (PAs) that control pulmonary arterial pressure. We recently showed that endothelial TRPV4 (transient receptor potential vanilloid 4) channels are key regulators of endothelium- dependent vasodilation in PAs. In this application, we provide novel preliminary data that endothelial TRPV4 channels regulate resting pulmonary arterial pressure (PAP), and are impaired in PAH. Moreover, we show that scaffolding protein caveolin-1 provides a signaling platform for protein kinase C (PKC)-dependent regulation of TRPV4 channels, a mechanism that is defective in PAH. Interestingly, two different peroxynitrite scavengers restored endothelial TRPV4 channel activity in PAH, suggesting that peroxynitrite may be a key contributor to dysfunction of TRPV4 channel function. Additionally, exogenous peroxynitrite also impaired caveolin-1PKC regulation of endothelial TRPV4 channel function. We, therefore, hypothesize that peroxynitrite-induced impairment in caveolin-1PKC regulation of TRPV4 channel function contributes to the loss of endothelium- dependent vasodilation in PAH. In Specific Aim 1, we will use PAs from endothelium-specific TRPV4 and caveolin-1 knockout mice and human lungs to define a novel caveolin-1PKCTRPV4 vasodilator signaling complex that controls endothelial regulation of PAP. In Specific Aim 2, we will test the hypothesis that elevated peroxynitrite levels disrupt caveolin-1PKCTRPV4 vasodilator signaling complex in PAH. We will also determine whether lowering peroxynitrite levels in PAH reduces PAP in a TRPV4-dependent manner. These studies will establish that abnormalities in endothelial TRPV4 channels in small PAs contribute to PAH, and lay the foundation for novel therapeutic strategies to rescue endothelial TRPV4 channel function.